The proposed research is directed at identifying DNA sequences, both within and surrounding the type I and type II chicken collagen genes, which are involved in either positive or negative regulation of these genes in differentiated fibroblasts and chondrocytes. The state of methylation of both type I and type II collagen genes will be reinvestigated using a modified gel transfer procedure which will permit detection of very small fragments. Changes in chromatin structure, seen as DNAse I hypersensitive sites, will be looked for in type I and type II collagen genes and flanking gene regions in both fibroblast and chondrocyte nuclei to determine whether changes, if they occur, are associated with expression. To identify sequences involved in regulation fibroblasts and chondrocytes will be transiently transformed with plasmids containing from 2000 to less than 100 bp of 5' flanking gene sequences of the pro Alpha1(I), pro Alpha2(I) and pro Alpha1(II) collagen genes placed 5' to genes whose expression can be readily tested at the protein and RNA level, such as SV40 large T and small t antigens. Analogous transient expression experiments will be carried out using collagen mimigenes in which the 5' flanking region and first three or more exons are ligated to part of the last intron, 3' most exon and 3' flanking gene region to determine if genomic regions and/or 3' flanking regions are involved in regulation. Once regulatory sequences are identified, it may be feasible to use them to isolate transacting factors involved in regulation. Obviously the failure to properly regulate the precisely timed sequential expression of type I and type II collagen genes would result in abnormal limb development.